DE69434024T2 - SURFACE PROTEINS EXPRESSED ON HUMAN CORTICAL THYMOCYTES AND THEIR USE - Google Patents

Description

The The present invention relates to a cell surface protein based on human cortical thymocytes and tumor cells are expressed from the hematopoietic system have emerged, and its use. More specifically, the present invention relates to a new single-chain Polypeptide glycoprotein (hereinafter referred to as "JL1") which has a molecular weight of about 120,000 daltons and the partial sequence: V (valine) -L (leucine) -P (proline) -S (serine) -V (valine) -F (phenylalanine) -C (cysteine) -A (alanine) -I ( Isoleucine) -T (threonine) has and exclusively on cortical thymocytes and on malignant cells of T-lymphoblastic Leukemia-, T-lymphoblastic lymphoma cells, where the lymphoma is cortical Thymocytes and malignant cells of about 50% of all Types of leukemia, by immunohistochemical and flow cytometric analyzes be identified, expressed, and its diagnostic and clinical application in leukemia and T-lymphoblastic lymphoma.

Of the human body shows specific reactions to foreign substances, which he after is exposed to childbirth, and T and B lymphocytes and antigen presenting Cells are involved in immune responses to the human body protect. Lymphocytes are the main component of lymphoid cells and have an important role in the specific immune response during the Circulation through blood and lymph. The main function of B lymphocytes it is antibodies to produce against foreign substances. T lymphocytes come in two types one of which has the task of providing the specific immune response support and the other task is to kill cells that are infected with pathogens are. The task of presenting antigen Cells are non-specifically enveloping and infecting antigens to process them by slicing them into little peptides and to provide their information to T lymphocytes.

T-cell development in the thymus brings a complex series of proliferation, differentiation and selection stages with it. T cells are hematopoietic Stem cells found in the embryonic liver and postnatal Bone marrow are produced. They migrate into the thymus, differentiate themselves and they mature and then they migrate into the blood vessels and become mature T cells. After rearrangement of the T cell receptor germline gene in the thymus, thymocytes express T cell receptor complexes on their cell surfaces. Of the Gene rearrangement process requires the enzymatic system including RAG-1 and RAG-2. As a result, in the course of a complex operation one extremely high T-cell receptor diversity can be generated. Still can not all generated T cell receptors have their corresponding functions in the periphery. Cells, the antigens produced by the major histocompatibility complex (MHC) Class I and Class II are provided, do not recognize can and cells that show a strong response to self-antigens away. These processes is called positive or negative selection. T cell receptors can be theirs perform corresponding functions, when they recognize a foreign antigen on their cell surface own MHC molecule is bound. This training process takes place in the thymus.

T cells express not only T cell receptors but also various cell surface proteins. These proteins are also important for T cell function. Most of these T cell surface proteins are expressed in a wide range of the T cell subpopulation. However, some of them are only expressed at a specific developmental stage, and these proteins can be used as T cell surface markers for the determination of T cell maturation stages. Mainly the classification system of Renherz et. al. used to determine the stages of differentiation of thymocytes, in which T-cell surface molecules are used as markers. According to this classification, thymocytes are early in (a) thymocytes, CD4 ^- / CD8 ^- double negative, (b) simple thymocytes, CD4 ⁺ / CD8 ⁺ double positive and (c) mature thymocytes, CD4 ⁺ or CD8 ⁺ single positive, classified. Early thymocytes express CD7, CD38 and CD71 proteins. They are the cells that newly arrive in the thymus from the bone marrow and actively share. Rearrangement of T cell receptor genes - TCRβ- occurs at this stage. Simple thymocytes occupy most of the thymus and express CD1, CD2, CD3, CD4, CD5, CD8, and the like, newly on the cell surface, and TCRα genes are rearranged. Thereafter, mature thymocytes express T cell receptors on the cell surfaces. Only some of them can survive and pass to the next stage, most of them die (Nikolis-Zugic, 1991, Immunol., Today, 12, 65-70). The cell surface molecules in T cells and thymocytes can be used to determine the stages of differentiation and to classify the subpopulations. In addition, they can be used to diagnose and treat tumors that have emerged from T cells. These cell surface proteins play an important role in the function and development of T cells.

Thymocytes should die unless the T cell receptor is suitable. Death is not simply a passive death, but an active death that involves certain proteins or the synthesis of certain metabolic Products (Rothenberg, 1990, Immunol., Today, 11, 116-119). This cell death may require specific stimulation signals from the outside. The cell surface proteins may be involved in the signaling pathway. However, the identification of cell surface proteins was not yet possible. If simple thymocytes express the specific proteins for the transmission of signals for the selection of T cell receptors and programmed cell death, these proteins would be the inherent proteins of that stage. Although some cortical thymocyte antigens such as CD1a, b and c are known to be expressed on the thymic cortex, they are also expressed on various cell types such as dendritic cells, brain astrocytes and B lymphocytes.

A The object of the present invention is a cell surface protein, expressed on human cortical thymocytes and tumor cells, which have emerged from the hematopoietic system, which a single-chain polypeptide glycoprotein is of molecular weight of about 120,000 daltons, a subsequence of which is as follows: V (valine) -L (leucine) -P (proline) S (serine) -V (Valine) -F (Phenylalanine) -C (Cysteine) -A (Alanine) -I (Isoleucine) T (Threonine), provide.

One second object of the present invention is a monoclonal antibody to provide that protein or a fragment thereof its antigenic recognition site, recognizes.

One The third object of the present invention is a monoclonal antibody to provide that protein or a fragment thereof its antigenic recognition site, recognizes the stage of development of cortical thymocytes and tumors arising from the hematopoietic System emerged to diagnose.

One Fourth object of the present invention is a monoclonal antibody to provide that protein or a fragment thereof its antigen recognition site, and recognizes the tumor cells that from the hematopoietic System emerged kills.

The The present invention expresses a cell surface protein JL-1 on human cortical thymocytes, ready. The above-mentioned cell surface protein is a single chain glycoprotein with a molecular weight of about 120,000 daltons.

The Protein of the present invention is applied to human corticals Thymocytes are expressed and also on T-lymphoblastic leukemia and Lymphoma cells expressed from human cortical thymocytes emerged, and on cells of about 50% of the various Types of leukemia.

In addition, the present invention provides a monoclonal antibody which recognizes the protein JL-1. He is preferably derived from the human or animal body. The present invention also includes portions of the antibody, including its antigenic recognition site (V _H and V _L ), and thus has the ability to specifically recognize the antigen. It may also include an antibody fragment such as F (ab ') 2, Fab and Fv. Preferably, the antibody fragment (Fv) comprises a single-chain polypeptide fragment of the antigen, a so-called single-chain Fv, which is prepared by inserting a linker peptide between two polypeptides, V _H and V _L , to increase its thermal stability. The antibody or fragment thereof as mentioned above may comprise a labeling substance selected from a group consisting of radioisotopes, fluorescent substances and coloring matter.

The The present invention also provides a method of manufacture of the aforementioned antibody or fragments thereof, and provide a cell containing the antibody or the fragment thereof, and a method of production this cell.

Further The present invention provides an in vitro method of diagnosis of T-cell leukemia and T-lymphoblastic Lymphoma using the above-mentioned antibody or fragment thereof ready.

Furthermore The present invention provides a drug for the treatment of leukemia and T-lymphoblastic lymphoma using the above mentioned antibody or fragments of it ready. Preferably, the antigenic recognition material includes a toxic protein that is selected from a group those made from radioisotopes, toxic chemicals, toxic proteins and anti-tumor agents.

The present invention provides a medicament for the treatment of leukemia and T-lymphoblasti g. lymphoma derived from the hematopoietic system by gene therapy using the JL-1 protein as the target substance and an antibody or fragment thereof recognizing JL-1 as a 'targeting material'.

It also puts the present invention provides a method for identifying the JL-1 protein ready, the the steps comprise: (a) preparing an antibody by the above mentioned breeding a cell and its purification from it; (b) collecting the antibody which exclusively reacted with human cortical thymocytes by in vitro immunohistochemical staining of the thymus with the antibody purified in step (a); (C) Collecting the antibody, which reacts only with the thymus, by in vitro immunohistochemical coloring of normal human tissue with that collected in step (b) Antibody; and (d) identification of the protein present in leukemia cells and T-lymphoblastic Lymphoma is expressed by immunohistochemical staining the antibody collected in step (c).

1 is a photograph of the immunohistochemical staining of the thymus by the supernatant of a hybridoma clone producing the anti-JL-1 antibody;

2 Figure 10 is a photograph of a 10% SDS-PAGE analysis of the resulting product and intermediates at each step of the anti-JL-1 antibody purification process.

3 Figure 4 is a photograph of the expression of CD4 and CD8 molecules on the cell surface of JL-1 positive thymocytes using bicolor FACS;

4 Figure 15 is a photograph of a 15% SDS-PAGE analysis of anti-JL-1 antibody digested with pepsin for various times at pHs of 3.5 and 3.8;

5 Figure 15 is a photograph of a 15% SDS-PAGE analysis of the anti-JL-1 antibody digested with papain; and

6 Figure 3 is a photograph of an SDS-PAGE analysis of the JL-1 protein immunoprecipitated with anti-JL-1 antibody conjugated beads following radioisotope labeling of the thymocytes.

The following specific examples of biologically active polypeptides are performing for the present invention, but they should by no means be construed as limiting the present invention Considered invention.

example 1

Around a specific cell surface protein on normal thymocytes were discovered after the following Example human thymocytes are administered to Balb / c mice for antibodies against to produce human thymocytes.

10 ⁷ human thymocytes were administered intraperitoneally and Balb / c mice were immunized for six weeks at two week intervals. The spleen of Balb / c mice was removed 3 days after the last administration to prepare a spleen cell suspension. Monoclonal antibodies were generated by fusing the spleen cells of Balb / c mice immunized with human thymocytes with SP2 / 0-Ag14 mouse myeloma cells resistant to 9-azaguanine. The cell fusion method followed the Köhler and Milstein method (Köhler and Milstein, 1975, Nature, 256, 495-497). Ten ⁸ spleen cells were fused with 10 ⁷ myeloma cells using 50% polyethylene glycol 4000. The cells were washed and resuspended in DEAE medium containing 20% bovine serum albumin, 100 μM hypoxanthine, 0.44 μM aminopterin and 16 μM thymidine (HAT medium). The cells were seeded into four 96-well plates and grown at 37 ° C in an incubator to provide 5% CO ₂ . When colonies had formed after two weeks, the supernatant was generated and the reactivity of the antibody was determined using enzyme linked enzyme adsorption (ELISA) and flow cytometry.

The well containing more than 10 ⁵ cells per well was scored as a positive group. The cells were removed from the well containing high-reactivity antibodies, and 0.5 cells per well were subcloned by limiting dilution test to prepare a stable hybridoma clone with high reactivity of the antibody. This hybridoma clone secreted antibody into the medium and the supernatant was saved for the next steps.

Example 2

To find a clone among the hybridoma clones generated in Example 1 which secretes antibodies recognizing the specific cell surface antigens on cortical thymocytes, using the supernatant of the hybridoma clone produced in Example 1, an immunoperoxidase stain was made to be 4 μm thick Layer of fresh tissue and paraffin-embedded tissue according to the avidin-biotin complex (ABC) method by means of the binding of avidin with biotin on a microscope slide. The supernatant of monoclonal cells was used as the primary antibody. The paraffin-embedded tissue was treated with normal mouse serum and allowed to stand for 1 hour to prevent nonspecific background staining after removal of the paraffin. After addition of a primary antibody, it was allowed to stand overnight and washed three times with PBS. Biotinylated goat anti-mouse immunoglobulin used as a secondary antibody was added. It was allowed to stand at room temperature for 1 hour and washed three times with phosphate buffered saline (PBS). Then streptavidin and horseradish peroxidase conjugate were added. The ABC kit made by Burlingame was used for staining. H ₂ O ₂ -Aminoethyl carbazole solution was added and it was then washed with PBS three times after a reaction time of 20 minutes. After covering with coverslip and 80% glycerol gelatin, it was viewed under the light microscope.

Hybridoma clonal lines producing specific antibodies to human cortical thymocytes were selected. One of the clones whose antibody recognized only cortical thymocytes was designated H-JL1. The antigen recognized by the anti-JL1 antibody was designated JL1 and the monoclonal antibody anti-JL1 antibody. 1 Figure 13 is a photograph of the immunohistochemical staining analysis of the thymus with the supernatant of the hybridoma clone producing anti-JL1 antibodies. As in 1 only the cortical thymus is positive and most of the cortical thymocytes are positively stained. The cell surface of the cortical thymocytes was heavily stained, suggesting that the antigen recognized by the anti-JL1 antibody is a cell surface antigen. Adrenal medullary thymocytes were not stained with the anti-JL1 antibody. This proves that JL1 is an antigen specific for cortical thymocytes.

Example 3

Ascites were prepared to obtain a high concentration of the antibody secreted by the hybridoma secreting anti-JL-1 antibody. Three weeks after 0.5 ml of pristane was intraperitoneally administered to Balb / c mice, 10 ⁷ H-JL-1 hybridoma clones were raised which were grown in DMEM containing 10% bovine serum albumin. After 2 to 3 weeks, the ascites were harvested. The concentration of antibody is then 5 to 10 mg / ml. Only the immunoglobulins that responded to human thymocytes were purified, as there are many contaminating proteins in the ascites, such as albumin. Q-Sepharose chromatography and hydroxyapatite (Bio-gel HTP gel, manufactured by Pharmacia) chromatography were performed to purify antibodies from ascites containing a high level of antibodies obtained from Balb / c mice which were monoclonal anti-HJL-1 hybridoma cells were administered intraperitoneally.

3.14 g of ammonium sulfate per 10 ml of ascites fluid were added slowly on ice (precipitated with 50% (NH ₄ ) ₂ SO ₄ )). The mixture was centrifuged at 15,000 rpm for 30 minutes, resuspended in deionized water and dialyzed in 1 liter of buffer solution (20 mM phosphate, pH 7.4). The solution was passed through a Q-Sepharose column previously equilibrated with buffer solution (20 mM phosphate, pH 7.4) and adsorbed thereto, and then buffer solution was again passed through the column to remove free proteins from the column. Thereafter, the protein bound to the column was measured by a linear gradient from 0 M to 0.8 M NaCl using buffer solution I (20 mM phosphate, pH 7.4) and buffer solution II (20 mM phosphate and 0.5 M NaCl, pH 7.4). Each fraction was subjected to SDS-PAGE electrophoresis and the fractions containing the anti-JL-1 antibody were collected.

The Fractions were then dialyzed and replaced with a buffer solution previously (20 mM phosphate, pH 6.8) Hydroxyapatite column run. The fraction (20 mM phosphate, pH 6.8), by the pillar was sent to remove free proteins by means of a linear gradient from 0 to 0.3 M phosphate using the buffer solution III (20 mM phosphate, pH 6.8) and buffer solution IV (300 mM phosphate, pH 6,8) elutes. Each fraction was subjected to SDS-PAGE and the fractions containing more than 95% anti-JL-1 antibody were collected. The collected anti-JL-1 antibodies were against suitable buffer solutions dialyzed and stored. Of 1 mg of ascites were repeated by repeated trial versions 5 to 10 mg of anti-JL-1 antibody produced.

The electrophoreses of the intermediate and the final product in 10% SDS-PAGE are recorded in 2 shown. The first lane is ascites, the second lane is the precipitate by 50% ammonium sulfate prior to Q-Sepharose chromatography, and the third and fourth lanes are some of the eluted fractions of the proteins bound to the Q-Sepharose column, the fifth lane is the fraction collected from the fractions which had many anti-JL-1 antibodies among the fractions of Q-Sepharose chromatography before hydroxyapatite column chromatography, and which are sixth, seventh, eighth, ninth, tenth and eleventh lanes some of the eluted fractions of the proteins bound to the hydroxyapatite column.

Example 4

The This example was prepared using the Example 3 purified anti-JL-1 antibody as primary antibody according to the immunochemical Test carried out in Example 2 to determine if the JL-1 antigen is in normal tissue except in the thymus is expressed. Table I below shows the distribution of the JL-1 antigen in each tissue. Except Thymocyten none of the including other tissues peripheral lymphoid tissue, cerebellum, pancreatic tissue, ovary and Testes, skin, lungs, adrenals and kidney after staining positive. This result confirms that JL-1 is an antigen specific for cortical thymocytes.

TABLE I

Example 5

Example 2 and Example 4 show that the JL-1 antigen was expressed only on cortical thymocytes. In this example, the JL-1 antigen on normal cells and on malignant hematopoietic cells was analyzed by flow cytometry analysis. 1 × 10 ⁶ cells were placed in a Falcon tube and centrifuged at 1500 rpm for 5 minutes, and the precipitate of 1 × 10 ⁶ cells per 100 μl was suspended in PBS. 100 μl of the suspension was dispensed into test tubes and 100 μl of the anti-JL-1 antibody solution containing 1 μg of the purified anti-JL-1 antibody was added and mixed. The solution was reacted for 30 minutes at 4 ° C, centrifuged for 5 minutes at 1500 rpm and the precipitate washed twice with PBS to remove the antibody that had not reacted. The precipitate was suspended in 50 μl of the solution containing the diluted secondary antibody (FITC conjugated goat an ti mouse Ig, manufactured by Zymed) and reacted in the darkroom for 30 minutes at 4 ° C. 150 μl PBS was added, centrifuged and the cells were washed twice with PBS. After centrifugation, 200 μl of PBS was finally added to the precipitate. The content of positive cells and the intensity of staining were analyzed by flow cytometry (FACSscan, manufactured and sold by Becton-Dickinson). Antibodies directly linked to FITC (fluorescence isothiocyanate) or PE (phytoerythrin) were used as anti-JL-1 antibodies. In this case, it is not necessary to use the secondary antibody for fluorescence. The result is shown in Table II.

The Table II below shows the expression of the JL-1 antigen the cell surface from normal cells, normal spleen cells, bone marrow cells, PBMC (peripheral mononuclear Blood cells), activated PBMC cultured in medium containing 10 μg / ml PHA (Phytohemagglutinin), 3 μg / ml PWM (pokeweed mitogen) and 0.5 μg / ml anti-CD3 antibody. All of these reacted negatively with the JL-1 antibody. A positive answer showed only on thymocytes and 80-90% of cells before and after at birth expressed the JL-1 antigen. That means that JL-1 antigen proved is an antigen which, as previously shown in Example 4, specific for Thymocytes is.

TABLE II

Example 6

JL-1 positive cells were purified using a JL-1 antibody, and a two-color FACS analysis was performed using an anti-DC4 antibody and an anti-CD8 antibody to determine when the JL-1 Antigen during thymic tonogenesis. The Analytical method was carried out according to the method described above by using FITC-conjugated anti-CD4 antibody as anti-CD4 antibody and PE-conjugated anti-CD8 antibody as anti-CD8 antibody. 3 is the result of a two-color FACS analysis showing the expression of CD4 and CD8 on JL-1-positive thymocytes purified by the panning method. It was demonstrated that more than 99% of JL-1 positive cells were dual positive CD4 and CD8 cells. That is, JL-1 positive cells were normal thymocytes.

The Marker analysis using the anti-JL-1 antibody is shown by Flow cytometry is a cell surface expression of the JL-1 antigen on leukemia cells. B lymphocytes, monocytes and tumor cells that have emerged from myelocytes are negative for the JL-1 antigen. T lymphocytes derived from normal thymocytes are positive for the JL-1 antigen and other T lymphocytic tumor cells were negative. However, as can be seen in Table III, leukemic cells were from 50-60% of different types of leukemia patients, positive for the JL-1 antigen (Table III). The JL-1 antigen and the anti-JL-1 antibody could therefore strong diagnostic and therapeutic tools for various Types of leukemia and T-lymphoblastic lymphoma.

TABLE III

Example 7

The present example was conducted to show that the specific immunohistochemical staining of the thymus in Example 4 and the specific fluorescence staining of tumor cells resulting from thymocytes in Example 5 and from the hematopoietic system in Example 6 are a result of specific binding by is the antigen-recognition region (V _H + V _L ) of the anti-JL-1 antibody, and not that of binding by the Fc receptor on the surface of tumor cells resulting from cortical thymocytes. To fulfill the purpose of this example, the Fc region of the antibody was determined by means of Protease was removed, the F (ab ') ₂ fragment and the Fab fragment were purified and immunohistochemical staining of the thymus and flow cytometry of thymocytes were performed. In general, when the antibody of a Balb / c mouse is of the IgG1 type and treated with papain, an approximately 50 KDa Fab fragment and Fc fragment were prepared and when treated with pepsin, approximately 102 KDa F (ab ' ) 2 and the Fc fragment was digested into small fragments.

10 mg of the anti-JL-1 antibody was dissolved in deionized water and dialyzed in 1 liter of buffer solution (0.1 M citrate, pH 3.5) to prepare F (ab ') _{2 of} the anti-JL-1 antibody , 0.1 to 0.2 mg of pepsin, manufactured and sold by Sigma, was added to the solution, and the extent of digestion was determined after a reaction at 31 ° C, the solution after sufficient reaction by addition of 1/10 volume was neutralized to buffer solution to terminate the digestion of proteins by pepsin. The solution was dialyzed in buffer solution (20 mM phosphate, pH 8.0) on Q-sepharose column previously equilibrated with buffer solution (20 mM phosphate, pH 8.0), and then the buffer solution (20 mM phosphate, pH 8.0) through the column to remove free proteins and then with a gradient from 0 M to 0.5 M NaCl using buffer solution I (20 mM phosphate, pH 8.0) and Each fraction was subjected to SDS-PAGE to collect the fractions containing the F (ab ') ₂ fragment collected F (ab ') ₂ fragment was dialyzed in the appropriate buffer solution and stored.

4 is the result of electrophoresis of the anti-JL-1 antibody digested with pepsin at 37 ° C in buffer solutions (0.1 M citrate) at pH 3.5 and 3.8 for various times. The first to fifth lanes are at pH 3.5 for 0, 0.5, 1, 2 and 4 hours, the sixth to tenth lanes are at pH 3.8 for 0, 0.5, 1, 2 and 4 hours and the eleventh lane are standard proteins of known molecular weights. The upper thick bands of the first and sixth lanes are anti-JL-1 antibody, and the thick band under the anti-JL-1 antibody band of the second to fifth lanes and the seventh to tenth lanes are the F (ab ') ₂ - Fragment with a molecular weight of 102 KDa.

10 mg of the anti-JL-1 antibody was dissolved in 5 ml of buffer solution (20 mM phosphate, pH 7 to 8) and dialyzed in 1 liter of buffer solution (20 mM phosphate, pH 7-8) to give Fab of the anti-JL antibody. 1 antibody produce. Cysteine and EDTA were added to the solution to give a 15 mM and 1 mM solution, 0.2 mg papain, manufactured and sold by Boehringer Mannheim, was added to the solution, and the extent of digestion after reaction at 37 ° C determined. The solution was neutralized after sufficient reaction by addition of 1/10 volume of 1 M iodoacetamide to terminate papain digestion of the proteins. The Fab fragment was purified according to the procedure for preparing F (ab ') ₂ . The fragment thus obtained was dialyzed in the appropriate buffer solution and stored.

5 is the result of electrophoresis of the anti-JL-1 antibody digested in a buffer solution (20 mM phosphate, pH 7.0) with papain at 37 ° C for various times. The first to fifth lanes are for 0, 0.5, 1, 2, and 6 hours, the sixth lane being standard proteins with molecular weights of 116, 85, 53, 39, 27, and 14 KDa, respectively, as seen from above. The upper thick band of the first lane was anti-JL-1 antibody and the thick band between 53 KDa and 39 KDa of the standard proteins was the 50 KDa Fab fragment.

The F (ab ') ₂ fragment and the Fab fragment purified in this manner were immunohistochemically used to stain the thymus according to Example 4, and a positive reaction was observed. The JL-1 antigen of tumor cells resulting from thymocytes and cortex-thymocytes was observed by flow cytometry according to the procedures of Example 5 and Example 6 and showed a positive reaction as in Example 5 and Example 6. That is, the recognition of cells expressing JL-1 on the cell surface by the anti-JL-1 antibody is due to the specific recognition by the antigen recognition region of the anti-JL-1 antibody, and not due to nonspecific binding of the anti-JL-1 antibody through the Fc receptor on the surface of tumor cells derived from cortex thymocytes via the Fc region. An anti-JL-1 antibody fragment having an antigen recognition site is as useful in detecting the JL-1 antigen as the anti-JL-1 antibody.

Example 8

The following in vitro tests were conducted to investigate the possibility of using the antibody against the JL-1 protein for the treatment of leukemias and lymphomas. The anti-JL-1 anti body in RPMI medium containing 30% pooled AB human blood serum without complement inactivation was added to 5x10 ⁵ T-lymphoblastic leukemia cells / ml, grown for 12 hours, and tumor cell survival using tryphan blue staining observed. This result strongly suggests that tumor cells can be killed when antibody to the JL-1 protein is administered to leukemia cells. The killing of tumor cells by the anti-JL-1 antibody is mediated by cell lysis, which is dependent on either the complement system or the Fc portion of the antibody. This process is considered to be the result of complement mediated lysis.

Example 9

Immunoprecipitation and SDS-PAGE were performed to examine the biochemical characteristics of the JL-1 protein. 1 × 10 ⁷ human thymocytes or Molt-4 tumor cells washed with PBS (phosphate buffer solution) were suspended in 100 ml PBS. 5 μl of lactoperoxidase and 250 μCi of Na ¹²⁵ I were added, H ₂ O ₂ was added and reacted for 3 minutes, and the reaction was washed with PBS. Cells whose cell surface was labeled with ¹²⁵ I were suspended in lysis buffer solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% w / v nonident P-40 and 1 mM PMSF) 1 × 10 ⁷ cells per 1 ml, and shaken at 4 ° C for 30 minutes. The lysate was centrifuged at 3,000 rpm for 7 minutes, the supernatant was retained and the precipitate discarded. The supernatant of the lysate was reacted with 20 μl of protein A-Sepharose CL-4B beads manufactured by BioRad for 2 hours and then reacted with protein A Sepharose beads conjugated to rabbit anti-mouse Ig to pre-remove nonspecific material. The remaining lysate was reacted with 20 μl of rabbit anti-mouse Ig protein A Sepharose beads conjugated to anti-JL-1 antibody for more than 12 hours. The precipitate was boiled in electrophoresis buffer solution (0.125 M Tris HCl, pH 6.8, 4% SDS, 20% glycerol and 1% β-mercaptoethanol) for 10 minutes to elute the antigen, and subjected to SDS-pAGE, to find the desired protein band. An 8% acrylamide gel was used for the electrophoresis, after which the gel was dried and Hyperfilm-MP, manufactured by Amersham, exposed for autoradiography at -70 ° C for 1 day.

6 Figure 4 shows an SDS-PAGE analysis of the JL-1 protein immunoprecipitated after the thymocytes were labeled with radioisotopes. The first lane was electrophoresis of the JL-1 protein produced by immunoprecipitation of the thymocyte lysate under non-reducing conditions and the second lane was electrophoresis under reducing conditions. A band having a molecular weight of 120,000 daltons appeared under both reducing and non-reducing conditions. That is, the JL-1 protein contains a single polypeptide chain. In addition, the band slowly migrated under reducing conditions, suggesting that a disulfide bond is present in the protein. Elektophoresis analysis after treatment of the JL-1 protein with endoglycosidase F resulted in faster migration of the protein, suggesting that JL-1 is a glycoprotein. Thus, the JL-1 protein is a glycoprotein that has a simple polypeptide chain and a molecular weight of 120,000 daltons. CD1a, CD1b and CD1c were known to be expressed only on cortical thymocytes during T-cell development. CD1a, however, is a heterodimer consisting of a 49,000 dalton molecular weight polypeptide and a polypeptide designated β2m having a molecular weight of 12,000 daltons. CD1b is a protein consisting of 45,000 dalton molecular weight polypeptides and β2m, and CD1c is a protein consisting of polypeptides of molecular weight 43,000 daltons and β2m. However, these proteins are different than the protein of the present invention. Further, CD1a occurs on the cell surface of dendritic skin cells, Langerhans cells and brain astrocytes, and CD1b occurs on the cell surface of dendritic skin cells, brain astrocytes and B lymphocytes, and CD 1 c occurs on dendritic skin cells and B lymphocytes. Purification of the JL-1 molecule was performed using an affinity column made from conjugation of 100 μg of purified anti-JL-1 antibody to Sepharose beads. About 20 μg of the antigen was collected by boiling the beads with PAGE sample buffer. This protein was digested with trypsin and a representative fraction was selected using HPLC. The sequencing was performed by the Edman degradation method, the sequences were as follows. V (valine) -L (leucine) -P (proline) S (serine) -V (Valine) -F (Phenylalanine) -C (Cysteine) -A (Alanine) -I (Isoleucine) T (Threonine). The JL-1 protein and anti-JL-1 antibody are therefore very useful for the diagnosis and treatment of leukemia and T-lymphoblastic lymphoma cells.

The JL-1 protein of the present invention is useful for the diagnosis of lymphoblastic T-cell lymphoma and T-cell leukemia by determining whether the JL-1 protein is expressed after examination of tissue or the examination of peripheral blood because the thymocyte-specific protein JL-1 is not found in normal tissue or activated peripheral blood except thymocytes. Paraffin-embedded tissue is used for the diagnosis of tumors. An antibody however, immature T cells that react with such paraffin-embedded tissue have not yet been found. JL-1 is stable in paraffin-embedded tissue, making it useful for the diagnosis of tumors. The antibody or ligand of the present invention is useful for the treatment of lymphoblastic T-cell lymphoma and T-cell leukemia in which JL-1 is expressed. The present antigen can be used as a target material for gene therapy because it is not expressed on normal tissue.

The mentioned above Description of exemplary embodiments biologically active Polypeptides is illustrative of the present invention. Because of the variations, for the expert it will not be obvious, however present invention to the individual ones described above embodiments to restrict. The scope of the invention is defined by the following claims.

Claims (21)

Cell surface protein expressed on human cortical thymocytes and tumor cells, which have emerged from the hematopoietic system, which a single-chain polypeptide glycoprotein is of molecular weight of about 120,000 daltons, a subsequence of which is as follows: V (valine) -L (leucine) -P (proline) S (serine) -V (Valine) -F (Phenylalanine) -C (Cysteine) -A (Alanine) -I (Isoleucine) T (Threonine). Cell surface protein according to claim 1, wherein the protein is based on leukemic cells of 50% of the leukemia patients is expressed. Cell surface protein according to claim 1 or 2, wherein the protein is on T-lymphoblastic Lymphoma cells expressed from human cortical thymocytes have emerged. Monoclonal antibody that repeats the protein Claim 1 or a fragment thereof comprising its antigen recognition site, recognizes. An antibody fragment according to claim 4, comprising the heavy chain variable region (V _H ) and the light chain variable region (V _L ) of the antibody of claim 4. The antibody fragment of claim 5, wherein the heavy chain variable region (V _H ) and the light chain variable region (V _L ) are linked together by a peptide. Antibody fragment according to claim 5 or 6, wherein the fragment is a toxic protein contains. Antibody fragment according to claim 7, wherein the fragment and the toxic protein are linked are to form a fusion protein. Antibody fragment according to claim 8, wherein the fusion protein is the structure and antigenic specificity of the antibody fragment according to claim 5. antibody according to one of the claims 4 to 9, wherein the antibody or the fragment is from a human or animal body. Cell for the preparation of the antibody according to claim 4 or of the antibody fragment according to claim 5 or 6. antibody or fragment according to any one of claims 4 to 6 or 10, wherein the antibody or the fragment comprises a marker. antibody or fragment according to claim 12, wherein the marker is a radioisotope, fluorescent material or dyeable material. In vitro method for determining the stage of development of cortical thymocytes using materials one of the claims 4 to 6 or 10. In vitro method for the diagnosis of leukemic Cells using materials according to any one of claims 4 to 6 or 10. In vitro method for the diagnosis of lymphoma cells using materials according to any one of claims 4 to 6 or 10. A pharmaceutical composition comprising the antibody or the fragment according to any one of claims 4 to 6 or 10. The pharmaceutical composition according to claim 17, wherein the antibody or the fragment for Cells comprises toxic material. The pharmaceutical composition of claim 18, wherein the toxic Material a toxic compound, a toxic protein, a radioisotope or an antitumor agent. Medicament according to one of claims 17 to 19 for the treatment of leukemia and T-lymphoblastic lymphoma. Method for identifying the protein after Claim 1, comprising the steps: (a) preparing an antibody by Breed the cell line of claim 11 and its purification therefrom; (B) Collecting the antibody, which reacts with cortical thymocytes by immunochemical in vitro coloring of the thymus with the antibody produced in step (a); (C) Collecting the antibody, which reacts only with thymus, by in vitro immunochemical staining of normal human tissue with the antibody collected in step (b) has been; and (d) identifying the protein present in leukemic Cells and T-lymphoblastic lymphoma is expressed with the in Step (c) collected antibodies.